Chromatographic separations of chemical or biological substance mixtures in liquid samples on chromatographic materials are usually carried out using columns in which the compressed packing of these materials is fixed by an adapter which may be stationarily set in position in the column tube.
The chromatographic materials may be elastic, quasi-elastic, or nonelastic. When nonelastic materials are used, after a certain period of time a defined packing of the material results inside the column due to sedimentation. Thus, it is not absolutely necessary to achieve physical compression of the chromatographic material upon insertion of the plunger.
The need for fixing the height and thickness of the packing results from the elastic behavior of certain chromatographic materials during packing under flow. As the result of sedimentation from homogeneous suspension, in particular highly porous carrier materials such as the known carbohydrate-or polymer-based gel-like matrices, for example Sepharose®, Toyopearl®, Fractogel®, Fractoprep®, Macroprep®, Unosphere®, and others, in addition to derivatives obtained from chemical surface modification, occupy a volume during the packing process which is dependent on the duration and linear flow rate. The packing pressure is ultimately the determining variable for the onset of compression of the gel. However, as soon as this pressure acting on the packed gel is discontinued, the elasticity of the packing becomes noticeable, such that an initially rapid and then slower expansion of the packed gel sediment occurs. This expansion comes to a standstill when the sedimenting effect of gravity is in equilibrium with the expansion pressure. As a rule, the volume resulting from the expansion of a gel suspension previously compressed under flow does not completely correspond to the volume obtained from the same quantity of gel as the result of sedimentation under the force of gravity.
Since the pressure drop over a given gel packing is a complex function of the linear flow rate, the viscosity of the eluent, the temperature, and other parameters, a characteristic compression results for gel packings operated in open mode, which in addition does not remain constant during a chromatographic separation. However, a fixed, defined packing density is absolutely essential for achieving reproducible results in chromatographic separations. For this reason, chromatographic columns to be filled with quasi-elastic carrier materials to a variably specified height are preferably equipped with adapters which seal the packing and which may be held in a defined position by mechanical locking, regardless of the pressure drop in the column. Such adapters generally require a complex design and contribute significantly to high manufacturing costs, in particular for small-volume columns. In another embodiment of such columns, the option of specifying an adjustable height of the gel packing is omitted in favor of a simpler design. A stationary end piece is used which a priori allows only a single specified bed height of the gel packing. For such columns, a particular difficulty of the packing procedure is closing off the column in the packed state of the chromatographic material without elastically re-expanding the gel in the process.
For operation, both types of columns are generally connected to a pump which delivers the eluent at a suitable flow rate and is able to overcome the pressure drop over the column. These columns must therefore be operated sequentially, even for small volumes of packed chromatographic material. In contrast, simultaneous operation of multiple columns requires the use of a corresponding number of pump systems, which entails a very complex equipment setup.
Specifically in the biological sciences, however, there is frequently a need for processing of numerous small-volume samples in chromatographic separation steps as simultaneously as possible. To solve the dilemma of either a high expenditure of time for sequential separations or the tremendous complexity of equipment for the simultaneous use of multiple chromatographic systems, various solutions have been developed in the past for which, however, either the actual chromatographic separation step is reduced to simple sequences of adsorption and desorption, or eluent no longer passes through the chromatographic medium in the sense of classical chromatographic columns. Therefore, these technical solutions for simultaneous separation of multiple samples do not permit separation results of a quality known to one skilled in the art from the classical chromatographic columns. This relates in particular to the achievable number of theoretical plates and to the asymmetry of the sample distribution in the chromatographic material. Mentioned as examples are “spin columns” (columns for centrifugation) and devices for carrying out batch adsorption (adsorption from a homogeneous suspension) in parallel using numerous samples. Spin columns, as disclosed in U.S. Pat. No. 6,103,195, for example, contain the particular chromatographic material as a filter cake, in a manner of speaking, which is held in the column by means of a filter plate. After it is loaded, sample liquid or eluent is caused to flow through the chromatographic material by centrifugation. Firstly, the chromatographic material, if it is one of the above-referenced quasi-elastic gels, is not present in a defined compressed packing. Secondly, the flow of eluent through the chromatographic material is basically asymmetrical in the centrifugal field, since, even in the most favorable case of a centrosymmetrical configuration of the column, the centrifugal force defines a right angle with respect to the path velocity only for the center longitudinal axis of the column, and for all other locations on the column cross section defines a different angle. As a result of the high mobility of the liquids applied, the chromatographic material has a nonuniform sample loading, and the subsequent elution also occurs in a nonuniform manner.
In other embodiments of columns which are to be operated simultaneously in large numbers without connection to pump systems, a uniform flow of the eluent through a usually cylindrical packing of chromatographic material occurs by the simultaneous application of vacuum to the discharge outlets of the columns. For this purpose, standardized systems of columns in the form of 96-well microtitier or filter plates are primarily used. Here as well, the chromatographic materials used are not present in a defined packed form. In addition, these systems are less well suited for applications involving proteins and other high-molecular biological substances, since after a period of time air or other gas passes through the chromatographic material. Although residual interstitial liquid may be removed from the chromatographic material by additional centrifugation, due to variable flow rates in vacuum operation in individual columns it is not possible to achieve uniform conditions for the separation processes. A further disadvantage of vacuum-operated multicolumn systems is the potential denaturing of proteins resulting from the formation of foam when the sample exits the chromatographic material, since the partial vacuum is particularly strong at this location.
One of the simplest options for simultaneously carrying out multiple chromatographic separations for biological water-soluble substances is the use of open gravity-operated columns. Such columns are available in many designs from numerous suppliers, and are provided for various purposes. Examples include the products Econo-Pac® columns or Poly-Prep® ion exchange columns (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) or Mobius® plasmid kits (Merck Biosciences GmbH, Schwalbach, Germany). These columns generally operate by means of gravity-driven stepwise elution, and allow true chromatographic separations to be carried out. However, in this case as well the chromatographic media are present only in a low packing density as a result of sedimentation of the gel particles under the flow rate induced by gravity. Thus, the efficiency of the separation media used, primarily with respect to the achievable plate number, is not fully utilized.
Separations carried out simultaneously in batch mode, i.e., by adsorption onto the separation medium from a homogeneous suspension and subsequent desorption, represent the simplest technique for simultaneous use of various chromatographic media with numerous samples. In principle, this technique only offers a separation efficiency which corresponds to one theoretical plate per separation step. The seamless continuity of successive settings of the adsorption equilibrium, such as that in a chromatographic column, is not provided. A further disadvantage of the batch method is the residual liquid in the intraporous volume of the separation medium in each step of the separation from supernatant, regardless of which method (decantation, filtration, etc.) is used for the separation. This intraporous liquid still contains all of the dissolved substances in the same concentrations as in the preceding work step, and thus transfers these to the subsequent step. In principle, therefore, in batch mode a result which corresponds to the chromatographic separation efficiency in columns is achievable only for a very large number of consecutively staged adsorption and desorption steps. Use of this method is therefore limited by obvious practical considerations.
From the standpoint of chromatographic separation efficiency, the technical solutions discussed have the disadvantage either that they may be parallelized only with a high expenditure of time and complexity of equipment, or that the chromatographic separation efficiency of the separation media cannot be fully utilized in the absence of sufficient and permanent compression of the media. The latter-referenced drawback in particular could be partially or completely eliminated by designing gravity-operated columns in which the chromatographic media are present with sufficiently dense packing. One skilled in the art is aware that as the compression increases in such a packing with the maximum possible homogeneity, such as that of bead-shaped microparticles, the volume present between the particles becomes smaller, thereby increasing the theoretical plate number of this packing in chromatographic separations. Thus, for a given separation medium the maximum plate number is achieved for a very specific optimal compression.
The technical object to be achieved, therefore, is to construct a chromatographic column having a simple design in such a way that for a uniform geometry of the column packing, various chromatographic media may be packed in the column at the respective optimal permanent compression. A further aim is to be able to simultaneously operate large numbers of the affected columns, under gravity alone or with a combination of the influence of gravity and centrifugal force, in applications involving stepwise elution. The affected columns may characteristically be manufactured economically in miniaturized form, and in their design for parallel use are compatible with existing laboratory robotic systems.